We have made substantial progress in understanding the role of the Drosophila MyoD homolog, nautilus, in myogenesis in the fly embryo. The highly organized and segmentally reiterated muscle pattern in the Drosophila embryo is prefigured by the arrangement of the founder myoblasts. The expression of nautilus, the only MyoD-related gene in Drosophila, is initiated at stage 9 in a subset of mesodermal cells that become incorporated into every somatic muscle in the embryo. At stage 11 the same cells begin to express the founder cell-specific marker, duf LacZ (rP298LacZ). We inactivated the nautilus gene using homology-directed gene targeting and inducible RNAi to determine if any aspect of founder cell function required nautilus gene activity. Both approaches result in a range of defects that include severe embryonic muscle disruption, reduced viability and female sterility, all of which are rescued by an hsp70-nautilus cDNA transgene. More importantly, the highly organized founder cell pattern needed to establish the proper muscle organization is disrupted to varying degrees in nautilus null embryos prior to MHC expression, and this prefigures the subsequent embryonic muscle defects observed at later stages in development. Both eve and tinman expression patterns are essentially normal in the nautilus null. Although nautilus does not specify the myogenic cell lineage, it has a cell autonomous role in establishing the correct muscle organization in the embryo through its regulation of the founder cell pattern. This work has been submitted to PNAS for consideration. Work is in progress to tag the endogenous nautilus gene for Chip on chip analysis of nautilus target genes and protein complexes during myogenesis. We have recently identified a novel miRNA that regulates nautilus and several mRNAs encoding proteins important for Drosophila myogenesis. In efforts to understand the molecular basis of RNAi and miRNA function in Drosophila we have reported on a novel mechanism that appears to involve an RNA-dependent RNA polymerase (RdRP) activity. siRNAs serve as primers to convert the target RNA into dsRNA which is then degraded by the RNase III-related enzymes, the Dicers, to produce new siRNAs while degrading the target RNA in the process. This result sheds light on the role of the siRNAs in RNAi and may explain the potentcy of the mechanism behind RNAi and post transcriptional gene silencing since very few molecules of dsRNA can inactivate 100-fold more of the cognate RNA. We have enriched the RdRP activity sufficiently to obtain mass spec data and we have confirmed the candidate protein is involved in siRNA function in RNAi. We have cloned the protein and we are testing its activity currently. This is a noncanonical RdRP and may be important not only for RNAi but also heterchromatin formation and maintainance. We have cloned Drosophila Dicers 1 and 2 and expressed the full-length cDNAs in baculovirus to produce active enzymes. Each is an RNase III with different properties that align one with the RNAi pathway and and the other in the miRNA pahtway. We have identified novel RNA-binding proteins that regulte Dicer activity in these pathways. We have started to explore the role of short RNAs in heterochromatin formation in Drosophila and have evidence that heterochromatic patches can be formed that require key proteins in the RNAi mechanism. This may reveal an entirely new approach to gene silencing that impact treatment of human disease in the future.